Abstract:

The invention reflects enhanced antibody expression of an antibody of
interest by cell lines transformed by random homozygous gene perturbation
methods to either increase or decrease the expression pattern of a gene
of the cell line other than the antibody of interest. The transformed
cell line exhibits specific productivity rates, SPR, for the RHGP
transformed cell liens of 1.5 or more, as compared with the antibody
expressing cell line parents prior to transformation by RHGP. A knock out
or anti-sense construct may be devised to reduce expression of the target
gene, a promoter may be inserter to enhance expression of the target
gene. The antibodies expressed by the transformed cell lines exhibit the
binding properties of their parent cell lines prior to transformation
with RHGP, and increase Total Volumetric Production of said antibody by
said cells in a given volume.

Claims:

1-8. (canceled)

9. A cell line which expresses an antibody of interest, said cell line
having been transformed by random homozygous gene perturbation (RHGP) to
alter the expression pattern of at least one gene of the genome of said
cell line other than a gene encoding said antibody through random RHGP to
either increase or decrease the level of expression of said one gene,
wherein said cell exhibits a higher specific productivity rate (SPR) for
said antibody higher than that exhibited by cells of said cell line
without having been transformed by RHGP.

10. The cell line of claim 9, wherein said cell exhibits an SPR for said
antibody that is at least 1.5 times higher than that of cells of said
cell line without having been transformed by RHGP.

11. The cell line of claim 10, wherein said cell exhibits an SPR for said
antibody that is at least 3.0 times higher than that of cells of said
cell line without having been transformed by RHGP.

12. The cell line of claim 9, wherein said expression pattern has been
altered to decrease expression of said at least one gene of the genome of
said cell line.

13. The cell line of claim 9, wherein said expression pattern has been
altered to increase expression of said at least one gene of the genome of
said cell line.

Description:

CROSS REFERENCE TO RELATED CASES

[0001]This application is a utility application claiming benefit of U.S.
provisional application Ser. No. 60/855,127, filed Oct. 30, 2006, which
is incorporated by reference in its entirety herein for all purposes.

BACKGROUND

[0003]1. Technical Field

[0004]The present invention relates to methods of altering cells to
enhance production of proteins they have been raised to express.
Particularly, this invention addresses the use of Random Homozygous Gene
Perturbation to enhance antibody expression of an antibody-expressing
host, by targeted insertion of DNA to either depress endogenous
expression of a host protein, or enhance expression of a poorly expressed
host protein, the change in expression being related to an increase in
expression of the antibody expressed by the host cell.

[0005]2. Background of the Technology

[0006]Antibodies, particularly monoclonal antibodies, have become
important biologic products both in mankind's arsenal against disease,
and in research and development. While not the "magic bullet" once
envisioned, more than a score of monoclonal antibodies, sometimes
referred to as mAb, have been approved for therapeutic use. Just a few of
these include the Trastuzumab antibody, the active agent in
Herceptin® approved for the treatment of some breast cancers,
Palivizumab, the mAb of Synagis® approved for the
prevention/treatment of RSV, and Bevacizumab, a mAb present in
Avastin®, approved for the treatment of colorectal cancer, and
indicated to be effective in treating other conditions. Many more are
known.

[0007]By contrast, there are literally thousands of antibodies, mAb and
polyclonal, employed as workhorses in laboratories and research
facilities around the world. Antibodies are useful as diagnostics, as
agents to bind and isolate target molecules, to differentiate cells for
testing, and other uses that take advantage of the specific binding
properties of IgG to select out a single antigen, typically a biological
molecule, bound or unbound, that may be of interest. Antibody production
is fundamental business.

[0008]Methods of making antibodies are well established, although
refinements are added constantly. The basic information was set forth as
early as 1975, Kohler & Milstein, Nature, 256: 495-497 (1975). To prepare
monoclonal antibodies, a host, typically a rabbit or the like, is
injected with the antigen against which a mAb is sought. Following
immunization, the spleen, and possibly lymph nodes, of the host are
removed and separated into single cells. These cells are then exposed to
the target antigen. Cells that express the desired mAb on their surface
will bind to the immobilized antigen. These cells are cultured and grown,
and fused with myeloma cells or other immortal cells to form hybridoma,
which can be cultured to recover the expressed antibody.

[0009]Most antibodies, and virtually all therapeutic antibodies, need to
be modified to avoid inducing a rejection reaction in a patient. The DNA
encoding the antibody expressed by the hybridoma is isolated, and can be
modified by the insertion or removal of bases, altered glycosylation
profiles, and manipulation of framework regions and complementary
determining regions, which affect the affinity and avidity with which the
antibody binds to its target antigen. The resulting antibodies are
humanized or "human" or otherwise modified (chimeric antibodies and
veneered antibodies are common in the art). The state of the art as of
about 1995 is reflected in U.S. Pat. No. 6,054,561, the relevant
disclosure of which is incorporated herein by reference.

[0010]Once prepared and isolated, the DNA encoding the antibody may be
transferred to a preferred mammalian cell line for expression in
"production" or commercial amounts. It has long been recognized that
Chinese Hamster Ovary cells (CHO cells) make excellent expression
vehicles for recombinant or non-endogenous DNA. See U.S. Pat. No.
4,816,567. There has been developed a series of DHFR deficient CHO cell
strains, which permit the amplification of inserted DNA encoding specific
proteins or DNA sequences, as set forth in U.S. Pat. No. 5,981,214. This
latter patent describes the use of homologous recombination to target a
specific gene or expression region of a cell--in the case in question, to
induce expression of a heterologous gene. Other suitable cell lines
include 293HEK cells, HeLa cells, COS cells, NIH3T3 cells, Jurkat Cells.,
NSO cells and HUVEC cells. Other mammalian cell lines suitable for the
expression of recombinant proteins have been identified in the
literature, and are equally suitable for use in the invention of this
application.

[0011]Once stabilized, current methods to increase production of the
valuable antibodies tend to focus on increases the total productivity,
that is, high volumetric productivity, so that a given amount of cells
produces a given amount of antibodies. These methods tend to focus on
improving the methods and environments used to cultivate the cells, to
enhance total antibody production. In general, antibody production of
greater than about 1 g/L is required for an industrially competitive
process. Individual CHO cells are typically expressing in the range of
10-15 pg/cell/day.

[0012]Homologous recombination has been used in many contexts since about
1985. It was originally employed as a "knock-out" tool, allowing the
suppression of an expressed gene, to study the response of the modified
cell. Subsequent procedures were developed to allow the silencing of
target genes. The use of anti-sense knock out constructs using a random
homozygous knock out method (RHKO) is described, e.g., in Li et al, Cell
85: 319-329 (196). In U.S. Patent Publication 20060240021 (U.S. patent
application Ser. No. 10/524,426 filed Aug. 18, 2003) the use of RHKO
techniques is disclosed to identify the genes involved in rapamycin
resistance. The entirety of that disclosure is incorporated herein by
reference. The ability to insert a construct into one allele, identify
the cells where that allele has been successfully modified by quick
throughput searching, such as for example by FACS (fluorescence activated
cell sorter) and similar methods makes this a superior technique for
selective identification and modification of a cell's genome. U.S. Pat.
No. 6,835,816, incorporated by reference herein discloses the use of this
technique in conjunction with genes reflecting tumor susceptibility,
including TSG101 genes.

[0013]Accordingly, it remains a goal of the industry to find a way to
increase the expression of antibodies, particularly recombinantly
prepared antibodies, from expression hosts like CHO cells, 293HEK cells,
HeLa cells, COS cells, NIH3T3 cells, Jurkat Cells, NSO cells and HUVEC
cells. and others, in a stable and reproducible fashion, using available
techniques to modify the genome of the cell.

SUMMARY

[0014]The invention demonstrates that cells that are good expression
vehicles for recombinant antibodies can be modified to increase the
specific productivity rate (SPR) of antibody producing cells by a factor
of 1.5, 2 or even 3 fold above the expression range capable of the cell
without such modification. Thus, by selectively altering the expression
profile of the cell, using knock out techniques (Random Homozygous Gene
Perturbation or RHGP) or expression enhancement techniques by inserting
expression promoters rather than anti-sense RNA or other expression
suppression constructs, antibody production by the cell can be enhanced.
Enhancement values of 3-fold or more, SPR, have been achieved by
suppression of the expression of targeted proteins. Enhanced SPR leads to
enhanced volume productivity, permitting commercial collection of mAb on
a heretofore desired but not achieved basis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a schematic illustration of the process of the invention.

[0016]FIG. 2 is a schematic illustration of the modification of a cell
line genome by random homozygous gene perturbation according to the
invention.

[0017]FIG. 3 is an illustration of the assays that can be used to
demonstrate enhanced antibody expression by cells transformed according
to the invention.

[0018]FIG. 4 is an illustration of how the repeated use of FACS sorting
assays can enable sequestration of the cells exhibiting the highest SPR
for a given antibody through the invention.

[0019]FIG. 5 is a schematic demonstrating the SPR enrichment for cell
lines transformed according to the invention using repeated FACS assays.

[0020]FIG. 6 is a graph showing the distribution of SPR for cells modified
by RHGP as compared with parent expression values.

[0022]FIG. 8 is a graph showing 3-fold enhancement of SPR and TPV using
the process of the invention.

[0023]FIG. 9 is a four part graph demonstrating correlation of SPR with
TVP of cells transformed with RHGP according to the invention.

[0024]FIG. 10 is a graph demonstrating the similarity in binding
properties of antibodies expressed by cells transformed by RHGP to
exhibit higher SPR values with parent cells of same cell line that did
not undergo RHGP.

[0025]FIG. 11 is a graph demonstrating long term stability of CHO cell
clones modified by RHGP to enhance antibody SPR.

[0026]FIG. 12 is a graph demonstrating the presence of elevated SPR and
TVP by several clones of a CHO cell line obtained by RHGP-induced
downregulation of Elmo1 expression.

[0027]FIG. 13 reflects the sequence for the Elmo1 gene of humans, mice,
rats and as present in CHO cells transformed by the invention.

[0028]FIG. 14 is a vector map of the plasmid used to induce downregulation
of the Elmo1 gene through RHGP according to the invention.

[0029]FIG. 15 is a blotting photomicrograph demonstrating downregulation
of Elmo1 in cells exhibiting enhance antibody production following
transformation by RHGP.

[0030]FIG. 16 is a graph demonstrating the increase in SPR of cells
modified by RHGP as compared with the decrease in expression of Elmo1.

[0031]FIG. 17 is a sequence comparison for the ion transporter protein of
human, rat, mouse and CHO cell, a target for RHGP pursuant to the
invention.

DETAILED DESCRIPTION

[0032]Applicants' invention resides in the discovery that the Specific
Productivity Rate or value of anti-body producing cells can be enhanced
by altering the expression profile of the cell's endogenous genome
without altering the genomic sequence about the antibody itself. Thus, as
noted above, it is possible to insert expression enhancers, amplifiable
genes, and the like, proximate to, or with, the inserted heterologous DNA
that expresses the mAb of interest. These methods have their limits.
Applicant's invention lies in the discovery that by inserting a construct
at a locus other than that which encodes the antibody itself, protein
expression profiles may be altered, thereby increasing he SPR for the
antibody. In many cases, this will involve introducing a knock-out
construct . . . and insert encoding, for example, anti-sense RNA, to down
regulate or suppress expression and even translation of a particular
protein. In other situations, it will involve inserting an expression
construct, or a construct involving an enhancer or promoter or some other
activator that enhances expression of a non-mAb protein, which is
implicated in the mAb synthesis pathway, and thus upregulates mAb
expression.

[0033]This is conveniently affected, in one example, by insertion of an
anti-sense knock-out construct that deactivates or inactivates an
unrelated protein. Not all knock-out or down regulation will increase mAb
expression. There does not appear to be at this time a way to map the
proteins whose expression profile can be affected in a way to predict
whether that alteration will increase SPR of a given cell. Predictably,
there are some proteins whose expression cannot be significantly
downregulated without adversely affecting survival of the cell. By the
same token, it is quite possible to increase expression of certain
proteins to the point where they are toxic to the cell. Applicants'
invention lies between these two extremes.

[0034]In general, there are two ways to improve antibody yield,
theoretically. One is to increase total productivity of a given quantity
of antibodies. There are limits on the improvements that can be made
without affecting the individual antibody-expressing cells. While one can
improve culture/fermentation conditions, improve spacing and the like,
real world limitations on the cost and capability of processing hardware,
the costs and frequency of media replacements, and the like combine to
limit the improvements available by manipulating the environment in which
the cells are grown to fractional or incremental improvements.

[0035]An alternative approach is to change the expression characteristics
of the cells themselves. If substantial improvements in cell SPR can be
made, without huge losses in volumetric productivity, and overall
increase in antibody yield is obtained. Applicants have discovered that
in fact SPR can be increased, as much as 300% or better, without a
concomitant loss in productivity of a given volume of cells, giving an
overall increase in antibody expression. Enhanced Antibody Production
(EAP) is thus achieved by insertion of a DNA construct at a locus distant
from the locus of the inserted antibody encoding sequence. This makes it
possible to increase the level of expression without endangering the
characteristics of the antibody itself or the insert region, which may be
critical to the expression of the heterologous antibody. Quality control
is satisfied by ensuring that the mAb products of cells exhibiting EAP
bind with the same relative avidity and affinity to the same target as
cells of the parent strain, before enhancement.

[0036]The process is generally indicated in FIG. 1, which constitutes a
kind of flow chart for the process of the invention. RGHP is used to
inactivate one gene per cell in a population of cells, thus creating a
RHGP library. The constituent cells of the library are subjected to a
high throughput assay system for the detection of enhanced IgG
production. The cells are altered using a Gene Search Vector (GSV) as
illustrated in FIG. 2. When integrated into an allele of the target cell,
the inserted construct is expressed--generating, in the embodiment
illustrated, an anti-sense RNA which effectively reduces expression of
the target protein. In alternative embodiments, the GSV may comprise a
sequence or fragment which boosts expression of the target protein.

[0037]The constituent members of the transformed library are then
subjected to a high throughput screening process, to identify candidates
exhibiting EAP. One assay in particular that lends itself to this process
is FACS. This is because transformed ells that express more antibody on
their surface will secrete or release more antibodies. Thus, a rapid and
high throughput low cost screening process selects out promising
candidates whose mAb expression level are higher due to transformation by
the GSV. To confirm that the high producers are in fact expressing the
antibody of interest, the pool selected is subjected to a conventional
ELISA assay, ensuring the antibodies secreted by the selected cells do in
fact bind to the target antigen.

[0038]It will be appreciated that many cells will respond to the initial
transformation by giving some gains in mAb SPR. To achieve the goals of
this invention, that is enhancing SPR by as much as 1.5 fold, all the way
up to 3-fold and beyond, only the most responsive transformants will be
selected. FACS screening, as described above, permits rapid
identification of EAP cells, in large amounts. This process is
illustrated in FIG. 4, where a first selection of, e.g., the top 5% (the
percentage collected will vary with the cell population, and it may be
anything from 25% down to 5%--representative values being between those
two endpoints, including 10, 15 and 20 percent by way of
exemplification). This "first cut is expanded, and subjected to a second
round of FACS sorting, again selecting a small percentage of the
antibody-expressing cells showing the highest SPR. This second collection
is then subjected to a third round, through single cell plating and
culturing conditions--yielding stable populations of antibody-expressing
cells exhibiting EAP and significantly higher SPRs than the original
parent strain prior to manipulation through RHGP.

[0039]As shown by actual example discussed, infra, involving decreased
expression of the Elmo1 gene, in fact, FACS can be used as described
above, to enhance antibody-production values, and SPRs, of RHGP
transformed cells. The repeated FACS selection "right-shifts" the
population of antibodies, with each sorting giving rise to a population
with a higher SPR--whether measured by mean, median or mode. The actual
utility of FACS sorting according to the invention is illustrated in FIG.
5.

[0040]Total volume productivity (TVP) screens are faster and easier to do
than selecting out individual improvements in SPR. Thus, the process can
be accelerated by taking a total productivity measure for all the members
of a transformed library. Since total productivity correlates with SPR,
by selecting out high productivity lines, likely sources of high mAb
expressing cell lines are the highest volume productivity cell lines.
Thus, FIG. 6 reflects an extinction experiment in which volume
productivity for an entire library of potential transformants is
measured, following RHGP. Thus, a number of cell lines actually show
inferior volume productivity, while the majority show at least some
degree of improvement, when compared with the non-transformed rent line.

[0041]The cell lines giving the highest volume productivity values from
the experiment reflected in FIG. 6 (this was done with the Elmo1
experiment set forth below--giving actual experimental values) were
measured for SPR as shown in FIG. 7, All but two of the cell lines giving
a higher total productivity on a 9-day extinction experiment gave SPR
values better than the parents--and as show, the parents were selected
for an already high SPR of 16 pg/cell/day. Cell lines expressing >50
pg/cell/day may be secured through this invention. This is illustrated in
FIG. 8, where at least one cell line, 296C2H, prepared by RHGP insertion
of the Elmo1 anti-sense RNA exhibited both SPR and volume productivity in
excess f this target value. All of the selected cell lines illustrated
show marked improvements in their SPR when compared to the high-producing
parent. Thus, given a simple transformation step well away from the cite
of the transforming antibody sequences, significant increases in antibody
expression are achieved. The correlation between SPR and total
productivity is also shown in FIG. 9, which shows growth kinetics for the
various cell lines. Depending on the envisaged facility and industrial or
commercial process, growth kinetics may impact the choice of the "best"
modified cell to select, given relatively similar TVP and SPR.

[0042]As noted above, it is important to develop a technique that is not
only simple, susceptible of application on a rapid throughput format, and
capable of giving substantial improvements in the SPR of a given
mAb-producing cell line, it is essential that the transformation take
place in a site remote from the antibody sequences themselves, so that
antibody properties are not disturbed. As shown in FIG. 10, the
antibodies of the RHGP transformed high SPR cells exhibit binding
characteristics not distinguishable from those of the parent strain. In
FIG. 10, the parent strain is given as the control. These increases are
stable over time. See FIG. 11. Equally important is the transformation
induced by RHGP pursuant to the invention results in stable increases in
SPR. As shown in FIG. 12, a number of clones from a single experiment
involving down regulation of the Elmo1 gene exhibited both higher SPR and
higher TVP.

Example 1

RHGP Using Antisense RNA of the Elmo1 Gene

[0043]The Elmo1 gene of C. elegans was identified as important in
phagocytosis of apoptotic cells, and for cell migration. Gumienny et al,
Cell. 107(1): 27-41 (2001). This gene was targeted with an anti-sense
knock-out RHGP, in an effort to improve higher antibody SPR in cells
expressing recombinant antibodies. The general strategy described above
was employed for this experiment.

[0044]When the individual phenotypes have been selected for cloning, the
target gene involved in enhanced antibody production was identified by
the strategy shown in FIG. 1. The vector map for the Elmo1 construct is
given in FIG. 14.

[0045]The full-length CHO ELMO1 cDNA was cloned into the expression
vectors of pCDNA3.1 and pLLexp with both orientations, which allow the
over-expression of the ELMO1 protein or production of the antisense RNA.
Since the anti-ITP antibody is not available, the CHO ITP cDNA was fused
with myc taq at its 5' or 3' end and cloned into pLLexp expression
vector. The fusion partner, myc taq will provide a domain for detection
for the expressed ITP protein level.

[0046]To verify that the phenotypes with higher SPR have the GSV insertion
in the genomes, the genomic DNA was first subjected to PCR amplification
of the chloramphenicol acetyltransferase (CAT) gene. Indeed, the PCR
analysis has indicated that all the single clones and the pools selected
by FACS have the CAT gene inserted in the genome. To identify the gene
involved in the phenotype of clone 296-C2H, the genomic DNA was digested
with restriction enzymes individually, which allow us to rescue the
genomic DNA along with the GSV. The digested genomic DNA was
re-circulated and used to transform E. coli competent cells. A total of
16-24 transformed colonies were picked for DNA preparation and sequencing
analysis with the LTR primers near the junctions between the GSV vector
and the genomic DNA. The regenerated genomic sequence was taken for Blast
Search in GeneBank. A 450-bp domain of CHO genomic DNA sequence shares
87% identities with the sequence on mouse chromosome 13, in which a gene
called engulfment and cell motility 1 protein (ELMO1) was located.
Especially, the further sequencing information revealed that the
corresponding exon 16 domain of CHO cells shares 95% homology with mouse
counterpart. Although the CHO genome sequence database is not available
in public databases, it's obvious that the GSV has been integrated in the
intron between the exon 15 and 16 in 296-C2H genome and interrupted the
ELMO1 gene according to the blast search information. The CMV promoter
from the GSV seems to transcribe the antisense RNA and knockdown the
ELMO1 gene in the phenotype, which has lead to the antibody production
enhancement. The ELMO1 gene has been identified from many other species,
such as mouse, rat and human, which has been reported to be involved in
the cells motility and required for cell phagocytosis and cells
migration. A 3.7-kb full-length ELMO1 cDNA was isolated from a CHO cDNA
library using a 31 nucleotide primer designed from exon 16 of CHO ELMO1.
The complete coding sequence of ELMO1 from CHO cells is 2181-bp long
encoding 727 amino acids protein. The CHO protein shares 99% homology
with mouse, rat and human homolog. (FIG. 13). The cDNA was then cloned in
pCDNA3.1 and pLLexp expression vector with both orientations for
validation of the gene in naive cell line (FIG. 14)

[0047]As discussed above, downregulation of the Elmo1 gene, following
insertion of the Elmo1 anti-sense "knockout" construct is correlated with
high SPR in RGHP clones from this experiment. See FIG. 15. Importantly
however, while some downregulation was observed, it was partial. Elmo-1
is still being produced, as would be expected, given the single allele
insertion. In contrast, the increase in SPR and TVP was profound. The two
correlated events, induced by a single round of RHGP followed by
selection as described above, are shown in a single frame in FIG. 16.

Example 2

Ion Transporter Protein

[0048]To demonstrate the efficacy of this invention, a second target for
RHGP was selected, this time an ion transport protein. What is of
fundamental importance is that this experiment demonstrates that proteins
can be downregulated (underexpressed as compared with the parent strain
expressing the antibody of interest) or upregulated (overexpressed as
compared with the unmodified parent strain expressing the antibody of
interest) and nonetheless give EAP. What is fundamentally important is
that the invention provides a method for modifying the expression pattern
of at least one protein of a genome, coupled with a facile method for
rapid detection and sequestration of cells expressing antibodies at a
significantly higher SPR than the parent cell line prior to
transformation by RHGP.

[0049]Using the same strategy, we have successfully identified the
insertion site of the GSV in the genome of another clone 263-C4G. The
genomic sequence contig was taken for Blast Search in GeneBank. The
genomic DNA sequence of 263-C4G shares significantly high homology with
that on mouse chromosome 13, in which the ion transporter protein gene
homolog (ITP) was located 15 kb downstream of the GSV insertion site.
Most likely, the CMV promoter of GSV has over-expressed the ITP homolog
and lead to the enhancement of antibody production in the phenotype.

[0050]The cDNA of ITP gene was isolated by RT-PCR with mRNA of 263-C4G.
The 2043-bp cDNA encodes 681 amino acids protein, which shares 96%
identities with rat, and 95% with mouse and human homolog (FIG. 17). The
ITP homolog belongs to the sugar-type transporter for the movement of
substances such as ions, small molecules and micromolecules.

Methods--Preparation of RNA and Genomic DNA.

[0051]The total RNA was isolated from CHO cells using TIRIZOL Reagent
(Invitrogen).

[0052]Following the manufacturer's protocol, 5-10×106 CHO cells
were used for each preparation. The mRNA was isolated using oligo dT
magnetic beads (Invitrogen). To isolate the genomic DNA, the CHO cells
(5-10×106 cells) were collected and washed once with PBS
solution. The cell pellet was resuspended in 10 ml of lysis buffer
containing 0.32 M Sucrose, 10 mM Tris pH 7.5, 5 mM MgCl2 and 1%
Triton X-100. The cell lysate was centrifuged at 1500×g for 15 min.
The supernatant was removed and the pellet was resuspended in 0.5 ml of
proteinase K buffer containing 25 mM EDTA, 150 mM NaCl and 40 mM Tris pH
7.5 and transferred to a 1.5-ml tube. Immediately, 10 μl of 10 mg/ml
proteinase K stock solution and 25 μl of 10% SDS were added to the
mixture. The solution was mixed gently and incubated at 37° C.
overnight. The next day, 5 μl of 10 mg/ml of RNAse A was added and
incubated at 37° C. for 2-4 hrs. After RNAse A digestion, the DNA
mixture was extracted twice with phenol/isoamyl alcohol/chloroform. The
DNA was then precipitated with equal volume of isopropanol and
centrifuged at 14000 rpm for 15 min. The pellet was washed with 70%
ethanol and dissolved in 200 μl of TE (pH 7.5) buffer. The DNA
concentration was determined by OD reading at A260.

Genomic DNA Cloning.

[0053]To identify the genomic DNA sequence surrounding the GSV insertion
site, 10 μg of each genomic DNA in 250 μl was digested with
restriction enzyme, such as BamHI and HindIII. The digested DNA was then
extracted once with phenol/isoamyl alcohol/chloroform and precipitated
with 2.5 volumes of ethanol. The DNA was air dried and dissolved in 30
μl of TE buffer. The digested DNA was then self-ligated with T4 ligase
at 16° C. overnight. The next day, the ligated DNA was
precipitated with ethanol and dissolved in 20 μl of TE buffer. The
ligated DNA was used for electroporation with ElectroMax DH10B competent
cells. Sixteen colonies from each ligated DNA were grown in 1.5 ml
culture for DNA preparation and digestion with the restriction enzyme for
size analysis. The plasmid DNA was further analyzed by DNA sequencing.

GenBank Blast Search and Genome Mapping.

[0054]The DNA sequences were taken for mouse genome homolog search through
NCBI Blast Search program. When the mouse homolog has been identified at
the insertion site, the genes in that locus surrounding the GSV could be
scanned and identified. The orientation of the CMV promoter in GSV will
decide either the gene has been knockdown or over-expressed by RHGP. If
there was no homology identified, the DNA sequencing will be continued
until the mouse homolog has been found.

Construction of the CHO cDNA Library.

[0055]The cDNA library was constructed with Invitrogen's SuperScript cDNA
System. Following the manufacturer's protocol, the synthesized double
stranded cDNA was ligated into a vector followed by transformation with
ElectroMax DH10B competent cells. Two million transformants from the
electroporation mixture were used to inoculate 100 ml of the TB broth
medium at 37° C. for overnight. The plasmid DNA of the library was
isolated with a Qiagen kit.

PCR Amplification of ITP cDNA.

[0056]Since the exon sequence of CHO ionic transporter protein is not
available, the target cDNA was amplified by PCR with degenerate primers
designed from the mouse ITP homolog. A 734-bp cDNA fragment in the middle
of the gene was first amplified with a pair of degenerate primers (L625:
5'AACGTGGTCAGCAARTGGGA3' and R1339: 5'TTCACYTCRTGGCCCATCAT3'). The
amplified cDNA fragment was completely sequenced. The 5' and 3' fragments
of the gene were subsequently amplified with the primers designed from
the known sequences of the internal fragment combined with the 5' and 3'
primers designed from the mouse ITP homolog. After the 5' and 3'
fragments of the gene were amplified and sequenced, the full-length ITP
cDNA was finally amplified by PCR with the primers designed from both
ends of the gene (ITP-L1: 5' CCCTGGCCATGGCGATAGAY 3' and C4G-R3: 5'
GGTCTGTAAACCTGTGTGCA 3').

[0057]While the present invention has been described with reference to the
specific embodiments thereof, it should be understood by those skilled in
the art that various changes may be made and equivalents may be
substituted without departing from the true spirit and scope of the
invention. In addition, many modifications may be made to adapt a
particular situation, material, composition of matter, process, process
step or steps, to the objective, spirit and scope of the present
invention. Of particular note is the fact that the expression pattern of
at least one gene of a genome of a cell line expressing an antibody of
interest is altered, followed by rapid screening to identify elevated
SPR. Identification of candidates offering EAP, in terms of both SPR and
TVP leads to expansion and stabilization of those cell lines using
standard procedure, as modified for each cell line type, and in light of
the modification leading to underexpression or overexpression of the
targeted gene. All such modifications are intended to be within the scope
of the claims appended hereto.